The challenges of setting standards for botanical identity testing.
On January 5 in France, the Directorate General of Competition, Consumption, and Fraud Repression (DGCCRF) declared that BL-DMAC, a botanical testing method, would be the only method approved for measuring the minimum cranberry proanthocyanidin concentrations required in order for a product to make a urinary tract infection health claim.
At first glance, this decisive step, albeit in only one country, sets a valuable precedent for the botanicals industry. It establishes a single, valid reference standard by which products can be compared and label claims made. In response to the BL-DMAC announcement, there was inevitable criticism-no botanical ID testing method is perfect, and none will achieve universal acceptance. However, the BL-DMAC case represents an interesting study of approaches to standardizing ID testing methods for botanical products.
While not insurmountable, the challenges industry faces regarding botanical ID testing, including developing standards for testing, are considerable. These challenges fall into at least three categories:
Scope: There are so many botanicals and so many active compounds to standardize that we encounter a Catch-22: the broader the standard developed, the less meaningful it becomes. Thus, a method like Folin-C for measuring total phenolics, while providing a starting spot for the antioxidant conversation, is too blunt an instrument for measuring antioxidants to meet most label claims. Is there a halfway point where established methods with valid standards might apply to a broader range of botanicals?
Precision: Can we get to a pure standard-or as close to 100% pure as possible? That is, can we ever achieve a standard that captures a 100%-accurate measurement of any botanical constituent? The industry has some standards that are close to being pure-for measuring certain catechin compounds, for example. But what about problem areas such as anthocyanins? These compound families are vital to the botanical industry, but while good HPLC testing methods are available for anthocyanins, good standards for testing are lacking.
Precision applies to both testing standards and methods, and precision can be lacking in both cases. For instance, standards may contain other, non-targeted substances, and testing methods generally do not extract all of the targeted compound from the sample.
Operating efficiencies: It is one thing to envision scientific solutions for ID testing according to standards; quite another to make those solutions cost- and process-efficient for industry. As industry sources contacted for this article point out, practical considerations such as the storage and degradation of reference standards play a major role in the success of any possible solution.
Ronald L. Prior, PhD, consultant and adjunct professor at the University of Arkansas (Fayetteville, AR), has conducted extensive research on botanical antioxidants. He describes the challenges of accurate ID testing, using the example of anthocyanins, one of industry’s primary targets of quality-control quantitation.
“Anthocyanins are relatively unstable molecules,” Prior says. “During processing or storage, particularly in solution, anthocyanins can react with other components present to form polymers. The term polymeric color has been used to describe these large-molecule compounds.” These polymeric colors may not have the same health benefits as the original monomeric anthocyanins, Prior explains. Depending on the method used for anthocyanin analysis, the presence of polymeric color may result in erroneously high readings for monomeric anthocyanins-giving a misleading indication of the level of the actual compounds present that are beneficial to health.
What would be on Prior’s wish list for developing better anthocyanins standards? “Cyanidin-3-glucoside is often used as a standard, but the ideal would be to have at least the glucoside form of all six aglycones-cyanidin, delphinidin, peonidin, pelargonidin, petunidin, and malvidin,” he says. He adds that bilberry, for instance, could be standardized using the six glucoside forms described above.
This gap between what is needed in high-quality standards versus what is currently available exemplifies the scope of the analytical challenge facing not only anthocyanins but other primary compound families as well.
The cost and time required to test according to a high-quality standard is also a concern. Mark Pedersen, ND, is vice president at Cornerstone Research and Development Inc., a specialized nutritional contract manufacturer in Ogden, UT. He summarizes a manufacturer’s primary concerns and challenges when considering testing methods and standards: “First are cost and method availability at any cost. Take bilberry, for example. There are at least four major anthocyanidins in bilberry, and sometimes to get a passing result, you have to standardize to all four peaks. That’s expensive [to do].”
“The second is precision. Methods rarely achieve 100% extraction capability,” he says, explaining that testing methods are rarely able to extract all of the target compounds from a botanical for testing. “That means we have to compensate, unless the customer allows a test range of, say, 90%. If we add a 15% overage, that [adds] costs, too.”
“The third is timing,” he concludes. “Time is money, and we would prefer testing turnaround time of five days, which matches our microtesting.”
Cal Bewicke, president of Ethical Naturals Inc. (San Anselmo, CA), a leader in scientifically standardized botanical extracts, echoes Pedersen’s concerns. “Phenolic compounds are a very broad category, and there are many individual phenolics. High-quality reference standards are generally available, but testing costs can get very high,” he says.
Luke Bucci, PhD, is vice president of research at Schiff Nutrition International (Salt Lake City, UT), a premium branded nutritional supplement company. Bucci enumerates a similar checklist of challenges encountered when developing testing standards: “1) getting a standard as close to 100% as possible on existing assays; 2) identification and validation of key compounds, including Standard Operating Procedures; 3) general acceptance by others in the industry (e.g., suppliers, regulatory agencies, competitors); and 4) cost containment.”
Bucci points to two additional factors that affect both the cost and performance of testing: “Storage of the standard to prevent water absorption and oxidation, and quantitation of undesirable compounds.”
From research to branded consumer products, there is strong agreement on some of the primary analytical challenges facing the industry.
Let’s return to the BL-DMAC case in France mentioned earlier. This situation exemplifies a kind of industry harmonization: a compound-specific method that received multilab validation and has been chosen by a government agency for the purpose of validating label claims. It suggests, at least, the following questions.
Is there a rationale for the need to develop product-specific methods, such as an A-type proanthocyanidin assay? Or, are broader measures more practical? The simple answer seems to be “Yes.” That is, there is a role for product-specific methods and for broader ones.
Bucci at Schiff, the brand product company, says that standards will be more useful if they have broader retail significance, while Bewicke at the ingredient company Ethical Naturals says that more-customized methods would be useful for big-impact ingredients with well-developed markets, like grape seed or green tea extracts. The economic value of these major ingredients makes the necessary R&D investment to develop ingredient-specific standards more attractive and feasible.
In either case, industry participants say, accuracy and validity are vital to a standard’s widespread use and acceptance.
Also something to consider: So far, we have been discussing methods for quantitating primary compounds. But what if we took a different approach? Although such quantitation methods may be necessary for label-claim compliance, perhaps we should look past quantitation methods to more-advanced performance methods.
Schiff Nutrition’s Bucci agrees that bioassays like ORAC need to be used more often. ”They are harder to validate, [but] they are more powerful than simple quantitation of individual actives and take into account both beneficial and harmful compounds.”
He explains why validation issues can be even greater for bioassays than for quantitative ones. Bioassays such as cell-based methods can provide a rich source of information about how substances behave in a live cellular environment. However, they more difficult to standardize than in vitro chemistry assays, precisely because they involve living cells.
With companies currently adhering to a range of different standards, Bewicke suggests that this may just lead to a lowest common denominator in testing. “Some companies work towards achieving the highest standards; others only wish to do the minimum to get by at the lowest possible cost. The end result in the products they offer is often very different,” Bewicke says.
The implication is that any uniform standard ought to have measurement thresholds that clearly delineate premium performance. This would seem to argue for methods that move toward efficacy or health outcomes and suggests the growing popularity of tests such as the cell-based Cardinal Wellness Factors from Brunswick Laboratories (Southborough, MA), which feature cellular antioxidant and anti-inflammation markers. These tests allow us to examine how substances with varying levels of, say, anthocyanins, behave in a cellular environment. They represent a natural and commercially important extension of testing, beyond standardized quantitation.
Whatever the method chosen, how do we approach what Bucci refers to as “universal agreement on assay methodology-or at least widespread use”?
An obvious answer is standardization by the industry’s institutional methods bodies-most notably, AOAC International and the U.S. Pharmacopeia (USP). Achieving this level of validation would certainly drive broad industry acceptance and use.
Schiff’s Bucci says, “AOAC is a good place to start, and USP is ultimately the way to go, but is very unwieldy. Perhaps more collaboration with FDA would help to develop a set of uniform standards-better if we show them how to do it right than for them to use a bad assay against us.” Bucci’s concerns are legitimate, as it can take years for method approval to wind through bureaucratic processes.
Cornerstone’s Pedersen reinforces both the merit and frustration in institutional method validation. “The FDA/AOAC and USP method-development committees can take up to five years to validate methods. Until then, it’s the Wild, Wild West.”
That is, if the methods are established at all. The Second International Conference on Antioxidants in 2005 concluded that an official method was needed for ORAC. AOAC, in its 2007 Executive Summary, listed ORAC as a “most likely to succeed” method. Yet, validation remains incomplete.
In the long run, Bewicke says, “A high-quality program of method validation, including written Standard Operating Procedures to minimize variability, is essential to producing results that are consistently accurate.”
One thing is clear-the industry won’t wait.
Based on our discussions, we have made a list of suggestions, in order of industry urgency:
1) Optimize efficiency of current analytical methods
2) Prioritize high-impact compounds for improved method development
3) Work toward industry-wide acceptance of best standards, where practicable
These suggestions are meant to be touchstones for discussion. Given the complex set of variables involved, we expect incremental progress. In the meantime, some advanced forms of testing, in lieu of industry consensus, will continue to separate the wheat from the chaff, botanically speaking.
Can Botanical Testing Standards Be Improved?
Ecuadorian Rainforest LLC: Steve Siegel, Vice President
"Ecuadorian Rainforest (ER) participates in a rigorous testing program. Using third-party laboratories, ER would fully support a botanical ID standard, within reason.
At the moment, ER performs the following tests on our products: Microbiological testing (Total plate count, yeast and mold, coliforms, E. coli, and Salmonella), heavy metals, moisture content, HPTLC, and HPLC, in addition to ID testing when available.
A universal testing program or method would be favorable but also seems a bit difficult, considering the many different biological factors of each ingredient. That, coupled with costs, would drive up the price of raw materials.
An issue ER faces is competition from Asia. Many products from that region try to “mimic” South American ingredients-for example, Brazilian acai versus Chinese acai. Many manufacturers are lured by low prices on products from Asia that are in fact not at all similar to the genuine ingredient that grows on the other side of the world. Proper identification would allow honest manufacturers to select ingredients that are genuine and of higher quality."
Nutraceuticals International: David Romeo, Managing Director
"One of the major issues with ID testing of botanical extract powders lies with the nonstandardized manufacturing processes used by the mainly overseas producers. When the whole botanical is turned into extract, the mixture of solvents that they may use to generate the liquid extract is highly variable. As such, the phytonutrients that are extracted and make up the signature of any finished product can also vary greatly.
For example, a manufacturer that uses predominantly water in a water-alcohol extraction will generate a product that is highly different from one that uses mostly alcohol in the same extraction technique. Both products can still be considered to be of the same origin and called an extract of botanical X; yet, the finished products may not have any fingerprint similarities due to the nature of the extraction process.
Now, if the lab performing the ID testing has no knowledge of the manufacturing process, then when they ID test against a whole reference botanical, they may well be looking for phytochemical components that may not be present, or may be present in an overwhelmingly concentrated form-either way suggesting a null-match.
For a universal set of ID testing procedures to be instituted, it is first necessary to have manufacturing standardized-or to have all botanicals/extracts bear greater details of manufacturing such that appropriate ID tests can be conducted by using comparators that are of the same nature (solvent extraction) as the test material."
Sabinsa Corp.: Benoit LeGall, QC Manager
"Sabinsa feels that there is a lack of quality standards as well as suitable methods for identification of botanical products.
Numerous botanical reference standards are simply not available in the market, and when they are commercially available, they are very expensive to purchase. Moreover, the U.S. regulators require the industry to carry out at least one ID test of botanical products, but there is no clear guidance on the type of test. The most common ID methods used by the industry vary, from macroscopic to microscopic evaluation (HPLC, HPTLC, GC, FT-IR, etc.).
Therefore, creating a uniform standard could be, at face value, a good idea. At this point, only the most common botanicals are described in official compendia and scientific publications. Considering the number of botanicals available in the market, the room for creating new standards and test methods, as well as improving the existing ones, is huge.
To be efficient, this important work requires close collaboration between the leaders of industry; public organizations, such as universities and standards-setting authorities (such as USP); as well as regulatory authorities."